An enzyme protein overexpressed in bacteria needs to be purified to show its activity. Why doesn't the cell lysate show activity when added to the substrate?
You need to check for incomplete lysis and also look for an efficient method of lysis which does'nt inactivate your protein.
If the enzyme is not expressed at a high level and its catalytic activity is also not very high in the assay being used, it may not be easy to detect its activity in the lysate.
The activity assay could also be problematic if the product to be measured is destroyed by another enzyme in the lysate.
In general, a cell extract is a mess... It may contain inhibitors of your enzyme, or other enzymes that consume the substrate and prevent detection of your enzyme's activity. Artifacts may also arise depending on the type of reaction you're trying to monitor and the assay you're using. If, say, your enzyme generates hydrogen peroxide you may not be able to detect this compound because some substances (thiols) in the cell extract readily react with it. All these problems, you don't have them with a purified enzyme. As the late Arthur Kornberg used to say "Never waste clean thinking on a dirty enzyme".
Due to so much gunk and excess endogenous DNA in cell lysates, you can't spectroscopically determine the concentration of your protein in the cell lysate. So you can measure activity but can't know what concentration of protein is producing that activity. Some investigators do gel filtration and collect fractions and do activity assays with the fractions to determine which fractions contain their protein of interest. Then they pool those fractions and further chromatographically purify their protein.
It saves you the trouble of having to do a western blot or mass spec analysis if your recombinant protein is inconveniently nearly exactly the mass of GroEL chaperone protein.
Hi Bin, the environment in a cell extract is not the same as the intracellular environment (which for example is heavily compartmentalized). But this is just a side point. If your enzyme shows activity when purified, but you can't measure any activity in cell extracts, then the bottom line is just one. There must be something in your extracts that interferes with the activity (or more likely the DETECTION OF activity) of the enzyme.
In my previous answer I provided only a few generic examples, because I don't have any information on your specific enzyme and on the assay you're using. You should be able to do better.
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